Download The hereditary pancreatitis gene maps to long arm of chromosome 7

 1996 Oxford University Press
Human Molecular Genetics, 1996, Vol. 5, No. 4
549–554
The hereditary pancreatitis gene maps to long arm of
chromosome 7
Louis Le Bodic1, Jean-Denis Bignon2, Odile Raguénès3, Bernard Mercier3,
Thierry Georgelin1, Mathieu Schnee1, François Soulard1, Katia Gagne2,
Françoise Bonneville2, Jean-Yves Muller2, Lucien Bachner4 and Claude Férec3,*
1Clinique
des Maladies de l’Appareil Digestif, Hôpital Laënnec, BP 1005, Nantes Cedex 01, France, 2Laboratoire
d′Histocompatibilité, ETS Loire-Atlantique/Vendée, BP 349, 44011 Nantes Cedex 01, France, 3Centre de
Biogénétique, Hospital, University, ETSBO, BP 454, 29275 Brest Cedex, France and 4GIS INFOBIOGEN, 7 rue
Guy Mocquet, BP8, 94801 Villejuif Cedex, France
Hereditary pancreatitis (HP) is an autosomal dominant
disorder with incomplete penetrance characterized by
recurring episodes of severe abdominal pain often
presenting in childhood. Although this disorder has
only been recently described, about 100 families have
been documented worldwide. The pathophysiology of
this disorder is unknown. Here, a large French family
of 147 individuals (47 of whom were affected) from a
four-generation kindred with HP has been examined
and a genome segregation analysis of highly informative microsatellite markers has been performed. Linkage has been found between HP and six chromosome
7q markers. Maximal two point lod scores between HP
and D7S 640, D7S 495, D7S 684, D7S 661, D7S 676 and
D7S 688 were 4.00 (θ = 0.143), 5.85 (θ = 0.143), 4.91 (θ
= 0.156), 8.58 (θ = 0.077), 8.28 (θ = 0.060), 4.40 (θ = 0.169),
respectively. Multipoint linkage data combined with recombinant haplotype analysis indicated that the most
likely order is : D7S 640 - D7S 495 - D7S 684 - D7S 661
- D7S 676 - D7S 688, with the HP gene situated in the underlined region. As in all families reported in the literature, the clinical presentation of the disease is identical
to the presentation of sporadic cases, one could expect that the knowledge of the HP gene could be a clue
to pancreatitis in general. Based on its map position,
this is the first step towards the positional cloning of
the Hereditary Pancreatitis Gene (HPG).
INTRODUCTION
Hereditary pancreatitis (HP) described by Comfort and Steinberg
in 1952 (1), is a form of chronic pancreatitis often present in
childhood. The first description of the disease, named by the
authors as hereditary chronic relapsing pancreatitis, was in a
family of which four members had a definite pancreatitis and two
others were probably also affected. Since the initial description
*To whom correspondence should be addressed
many other kindreds have been reported in different countries all
over the world (2–5). Most of the families reported so far are of
caucasian origin, although two families have been reported from
Japan (6,7). Hereditary pancreatitis is indistinguishable from
pancreatitis due to other causes. The disease is characterized by
recurrent episodes of pancreatitis from childhood, equal sex
distribution, a family history with at least two other affected
members and the frequent presence of calcified stones in
pancreatic ducts (8,9). The reports in the literature of about 100
kindreds and of at least three families with identical affected
twins, strongly suggested the inheritance of the disease to be an
autosomal dominant one with incomplete penetrance (10–13).
The pathogenesis of hereditary pancreatitis is not well understood
and the basic defect has yet to be identified. The first step to
improve our understanding of the disease is to map and to clone
the HP gene with the aim of identifying the gene as well as its
protein product. We have studied for many years one of the largest
kindreds reported so far with more than 220 individuals spanning
five generations, and have recently launched a systematic whole
genome search by linkage analysis (14). However as the major
constituent of pancreatic stones in HP is the pancreatic stone
protein, encoded by the ‘reg’ gene on 2p2, this gene is a potential
candidate for HP (15). Therefore as a first step, we looked for
linkage on chromosome 2. However, the lod score results were
consistently negative and this chromosome was excluded. The
subsequent strategy to map this gene was a genome wide search
by linkage analysis using sequence repeat polymorphisms. By
studying a large four generation French family with 47 affected
patients, we report in this paper the linkage data that map the gene
responsible for the phenotype of hereditary pancreatitis to the
long arm of chromosome 7 at 7q33-qter.
RESULTS
Description of the family
In 1962, Cornet et al. reported the first description of this family
including a cluster of 17 related patients with HP from Vendée
(western part of France) (16). Over the past 30 years different
genealogical studies led us to establish the pedigree described in
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Received November 29, 1995; Revised and Accepted January 26, 1996
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Figure 1. Pedigree of the family B.
Systematic screening of the genome
Using 213 microsatellite markers spaced at intervals of about 20
cM, with heterozygosities greater than 0.7, linkage analysis
allowed us to exclude half of the genome. Chromosomes 1, 2, 3,
9, 15, 16, 17, 19, 20 and 22 were scanned first as suggested by
Antonorakis (17). Then we screened the distal part of other
chromosomes. The first marker to demonstrate a lod score greater
than 3 was D7S 640 (lod = 4.00 at θ = 0.143).
Refined map of the distal part of chromosome 7
To determine the position of the HP gene relative to the
microsatellite markers in this region, 14 other markers were
examined (data not shown). Among these, lod scores greater than
3 were obtained with six markers. The greatest values were
obtained at D7S 661: lod = 8.58 at θ = 0.077; and at D7S 676: lod
8.28 at θ = 0.060. Two point lod scores obtained between eight
chromosome 7 markers and HP are shown in Table 1. Markers are
ordered from centromere (top) to telomere (bottom), inter-marker
distances (cM) are shown between them.
In order to determine the position of HP relative to the markers
used for the two point analysis, the location score was determined
by testing the locus of the disease and the markers.
Multipoint linkage analysis
As shown on Table 1, six chromosome 7 polymorphic markers
gave lod scores above the threshold value of +3, thus clearly
demonstrating linkage to HP.
Table 1. Results of two-point linkage analysis of family B
Locus
Zmax
θ Zmax
1.48
4.00
0.143
4.35
2.29
5.85
0.143
4.78
3.80
2.09
4.91
0.156
8.52
7.37
5.39
2.77
8.58
0.077
8.27
8.10
6.84
4.93
2.49
8.28
0.060
–0.38
2.50
3.92
4.33
3.36
1.61
4.40
0.169
–11.55
–7.35
–3.01
–0.59
1.29
1.61
1.04
1.63
0.280
–1.96
–1.37
–0.07
0.53
0.96
0.94
0.60
0.99
0.240
Lod score (θ)
D7S 640
0.00
0.001
0.01
0.05
0.1
0.2
0.3
0.4
–3.92
–1.36
0.64
3.02
3.85
3.83
2.92
0.78
0.96
2.47
4.63
5.66
5.61
–8.69
–1.20
0.80
3.50
4.63
6.00
6.01
6.97
8.46
3.33
5.56
7.40
–6.24
–3.31
–13.96
–1.98
7
D7S 495
4
D7S 684
8
D7S 661
1
D7S 676
3
D7S 688
2
D7S 505
2
D7S 642
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Figure 1. Of the 227 individuals, 214 are alive, 147 were studied
(47 affected) and these were only included after informed consent
was obtained. All the patients were evaluated by a gastro-enterologist (LLB, TG, MS, FS) (see Fig. 1: pedigree of family B).
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In order to localize more accurately the disease locus, a linkmap
analysis between the published map of these markers and HP was
performed. A composite map was generated by moving a four
marker window along the map and by determining the lod scores
(five points) for HP, for five equally spaced positions in the central
interval. This approach was adopted as it was impossible to run
a linkmap analysis with more than five points. Results were
compatible with those obtained with the various combinations of
four markers (from the eight used: not shown). We also checked
that the order of markers determined in our family by the various
five point ilink analysis was the same as the published order with
only slight differences in some distances (18).
The results in Figure 2 show that the most likely HP gene
localisations are proximal to D7S 640 at θ = 0.15 or between D7S
684 and D7S 661 at θ = 0.028 from D7S 684. Positions between
D7S 661 and D7S 688 cannot be excluded because the odds
against this were around 10.
Haplotype analysis
We performed haplotype analysis with the markers listed in
Table 1. This analysis revealed several recombination events.
Two recombination events in part 1 of family B (Fig. 3) were
observed between markers D7S 684 and D7S 661 both in subjects
GVI8 and GVI10 and one event was found in part 2 of family B
(Fig. 4) between markers D7S 676 and D7S 688 (individual
GVI23). The two recombination events in part 1 of family B place
the locus distal to D7S 684 and the recombination event in family
2 places the HP gene proximal to D7S 688. Individual GVI25 is
carrying the haplotype 1122221 linked to the disease but is still
not affected. We have observed two other individuals, VI28 and
his daughter (VII57) who are carrying the ‘disease haplotype’ but
are also still asymptomatic, this could be due to incomplete
penetrance of the disease. We have assumed the penetrance to be
80% for people over 20 and 65% for those under 20.
DISCUSSION
Over the past few years a very high quality short tandem repeat
polymorphism map has been generated by Weissenbach and
colleagues (19,20). These dinucleotide markers, which are highly
polymorphic have increased the efficiency of linkage mapping in
recent years, as further illustrated in this paper. Using this strategy
we have mapped the gene responsible for hereditary pancreatitis
to 7q. The markers were of (CA)n repeats and were chosen
because of their high heterozygosity (over 70%) and spacing of
about 20 cM. To facilitate the microsatellite genotyping, we used
a non radio-active multiplex procedure (21).
During our systematic screening effort to map the HP gene, we
followed the recommendation of Antonarakis who proposed the
use of an ‘intelligent linkage scanning’ technique (17). This
approach is based on the fact that the locations of genes already
cloned in the genome are non random; chromosomes which are
G+C rich have the highest densities of coding sequences, which
correspond to the CpG islands which are present at the 5′ end of
the majority of housekeeping genes. Chromosomes 1, 9, 15, 16,
17, 19, 20, 22 contain the highest densities of CpG islands, as do
the majority of telomeric bands. Following this strategy, we have
successively excluded chromosomes 1, 9, 15, 16, 17, 19, 20, 22;
following this we systematically screened the distal part of other
chromosomes (4, 6 and 7).
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Figure 2. Multipoint linkage analysis between HPG locus and markers D7S 640, D7S 495, D7S 684, D7S 661, D7S 676, D7S 688, D7S 505, D7S 642. Lod score
(base 10) for HP versus marker map (cM). D7S640 is at distance 0, marker positions are shown by a little triangle. This composite map was generated with overlapping
five point linkmap analyses, due to computation limitations. Kosambi mapping function was used.
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Figure 4. Pedigree and haplotype of parts 2 of Family B. The markers used for haplotype construction are shown, the cross-hatched bars represent haplotypes which
co-segregate with the disease, NA = not amplified fragment.
The first linkage obtained was to marker D7S 640, on
chromosome 7, subsequently, the marker D7S 661 also showed
very strong linkage with a lod score of 8.58 at θ = 0.077.
To refine the localization of the HP gene, polymorphic markers
proximal and distal to D7S 661 were studied and a multi-point
analysis was performed. Multipoint linkage data indicate that
the most likely order is: D7S 640 - D7S 495 - D7S 684 D7S 661 -D7S 676 - D7S 688, with the HP gene situated in the
underlined region.
Haplotype analysis and studies of crossovers show that the HP
gene is located between DS7684 and DS7688. Multipoint linkage
data indicate that the most likely position is distal to DS7684 or
between DS7661 and DS7676 as between these two markers no
recombination events were observed.
Our study maps the gene responsible for HP to 7q33qter. It also
illustrates the validity of the approach proposed by Antonarakis,
as HP gene was in fact mapped to a CpG island at the distal
telomeric part of chromosome 7. Definition of the haplotype
linked to the HP gene would allow presymptomatic diagnosis to
be performed in this family and in other families found to be
linked to chromosome 7q. This could be important for genetic
counselling in these families.
At the present time, it is difficult to speculate whether HP is a
homogeneous genetic disease or whether other families have
markers linked to the 7q locus. However, the general description
of families in the literature seems to be quite similar and
stereotyped. Linkage analysis performed in other families will
yield information about the genetic homogeneity or heterogeneity
of the disease. Clinically there are no clear definite criteria to
distinguish hereditary pancreatitis from idiopathic relapsing
pancreatitis. Both show similar symptoms. We are currently
collecting other pedigrees with HP to try to determine if other loci
for this disorder exist. The rapid development of improved
genetic maps as well as localization of expressed sequence tags
(22) would facilitate the search for candidate genes. Today it is
interesting to note that, among other genes, the carboxypeptidase
A has been mapped to 7q32-q ter which is close to our critical
region. Carboxypeptidase A is a pancreatic exopeptidase, assigned by Honey (23) to 7q32-q ter. As soon as mutations in the
gene causing HP have been identified, the next logical step will
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Figure 3. Pedigree and haplotype of parts 1 of Family B. The markers used for haplotype construction are shown, the cross-hatched bars represent haplotypes which
co-segregate with the disease, NA = not amplified fragment.
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be to study patients with sporadic pancreatitis. This may lead to
a greater understanding of pancreatitis in general (24,25).
Our locus assignment represents the first step towards the
identification of the gene responsible for hereditary pancreatitis;
identification of gene defects will contribute to, and improve our
understanding of the molecular basis of this disease.
553
ACKNOWLEDGEMENTS
MATERIALS AND METHODS
This work was supported in part by the Association Française de
Lutte contre les Myopathies (AFM), the Groupe de Recherche et
d’Etude des Génomes (GREG), bourse du Fonds de Recherche de
la Société Nationale Française de Gastro-Entérologie and Contrat de
Recherche Inserm. We thank I. Le Gall, J. Dausset and J.C. Dagorn
for their support, and Chantal Baclet for her secretarial assistance.
Linkage analysis
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Family studies
Each family member was examined by one of the clinicians
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and living participants gave informed consent. The diagnosis
criteria were those proposed by Gross et al. (4). An acute episode
of pancreatitis is the classical clinical presentation that is
indistinguishable from pancreatitis due to any other cause; that is
severe epigastring pain radiating to the back, which is alleviated
in the knee-chest foetal position. Symptoms in this family start as
early as 2 or 3 years of age for some children, and recurrences are
frequent. Calcifications of the pancreatic gland can be demonstrated by plains films of the abdomen and ultrasonography which
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EDTA blood samples (20 ml) were collected and DNA prepared
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The Fastlink package of programs, version 2,3P (26,27) was used
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noticeable changes in results.
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